Uptake by a specific target cell is a critical first step in gene transfer, which in most systems is limited by the endogenous entry pathway used by a particular gene transfer vector/virus. We have successfully engineered the Ad 5 vector to bind to a target cell through a different high affinity receptor than that normally used by the sub- group C viruses. Although we have made considerable progress in this area, there is still a considerable need for continuing our efforts to target Ad vectors. This is especially true if we are to target tissues such as well differentiated airway epithelium which have been reported to lack the high affinity as well as low affinity receptors that are used by subgroup C viruses for entry. Steps essential to Ad-mediated gene transfer: 1) attachment to the cell via the high affinity receptor (fiber binding to CAR, MHC-1), 2) facilitated internalization mediated by penton interaction with the alphavbeta3,5 integrins, and 3) endosomal escape which is a function of conformational changes in the major capsid protein hexon, are the aspects of Ad vectors which make them the most efficient gene transfer vector available. These proteins are also the primary targets of innate and acquired immune systems which are responsible for the lack of persistence of gene transfer by Ad vectors as well as the neutralizing immunity which compromises the effectiveness of repeat administration of Ad vectors. The focus of this project is to genetically modify the major capsid proteins of Adenovirus (Ad): hexon, fiber, and penton to the advantage of gene transfer of gene transfer to airway epithelial cells. It is our position that these proteins are the key mediators of both positive and negative attributes of Ad gene transfer vectors and our ability to genetically manipulate to genetically manipulate them will result in more efficient gene transfer, a greater degree of target cell specificity and finally a decreased in imunogenicity. Developing the capacity to modify the capsid proteins in our vectors will have direct application to any Ad vector system, including the "gutless vectors", chimeric virus vectors, and Ad vectors used to piggyback large DNA molecules.